Eiley Crenshaw

Plants, like other organisms, keep track of time through an endogenous circadian clock that is set by environmental cues. Distinct tissues exhibit varied clock activities; for instance, guard cells, pores on the leaf surface that promote gas exchange, demonstrate a longer circadian period compared to the surrounding mesophyll tissues. My aim is to determine if clocks from distinct tissues with normal circadian rhythms can communicate with and rescue disrupted clocks from similar or different tissue types. I hypothesize that wild type vascular clocks will be able to rescue the circadian rhythm of both disrupted mesophyll clocks and disrupted guard cell clocks. In order to determine the hierarchy of tissue-specific circadian clocks, I will mix different concentrations of protoplasts from mutant lines with Luc-reporter and wildtype protoplasts. Using protoplasts allows us to isolate and image cells from specific tissues. By combining protoplasts of different tissue types from wild type and mutant lines, my aim is to explore hierarchical aspects of communication between distinct tissues. I have optimized protoplast isolation from three major leaf tissues: mesophyll, vasculature and guard cells. In order to enrich guard cell protoplasts I am using D6:GFP lines to determine the purity of isolated protoplasts as they express guard cell specific GFP proteins. By measuring the luciferase signal with a CCD camera over a period of time I will be able to assess whether wildtype vasculature protoplasts are able to rescue the rhythm of short and long period mutants. While we know that plants do not have a centralized clock, this project will help us determine if there is a higher organization of the circadian clock in plant tissues. This study will help us understand the inter-tissue communication between different tissues that regulates the circadian clock.